DE3587697T4 - Cloning and expression of HTLV-III DNA. - Google Patents

Abstract

The determination of the nucleotide sequence of HTLV-III DNA; identification, isolation and expression of HTLV-III sequences which encode immunoreactive polypeptides by recombinant DNA methods and production of viral RNA are disclosed. Such polypeptides can be employed in immunoassays to detect HTLV-III.

Description

Technical areas

This invention is in the field of molecular biology and virology and particularly relates to human T-cell leukemia virus type III (HTLV-III).

background

The term human T-cell leukemia lymphoma virus (HTLV) refers to a unique family of T-cell trophic retroviruses. These viruses play an important role in the pathogenesis of certain T-cell neoplasms. At present, three HTLV types are known. A subgroup of the family, HTLV type I (HTLV-I) is related to the cause of adult T-cell leukemia lymphomas (ATLL) that occur in certain regions of Japan, the Caribbean, and Africa. HTLV type II (HTLV-II) has been isolated from a patient with a T-cell variant of hairy cell leukemia. M. Popovic et al., Detection, Isolation and Continuous Production of Cytopathic Retroviruses (HTLV-III) from Patients with AIDS and Pre-AIDS. Science, 224 : 497-500 (1984)

HTLV type III (HTLV-III) has been isolated from many patients with acquired immunodeficiency syndrome (AIDS). HTLV-III refers to the prototype virus isolated from AIDS patients. Groups reported to be at high risk for AIDS include homosexual or bisexual men; intravenous drug users; and Haitian immigrants to the United States. Hemophiliacs who have pooled People receiving blood products from donors and recipients of numerous blood transfusions are also at risk. The clinical manifestations of AIDS include severe, unexplained immunodeficiency, usually characterized by a decrease in T-helper lymphocytes. These may be accompanied by malignancies and infections. The mortality rate for patients with AIDS is high. A less severe form of AIDS also exists in which lymphadenopathy and decreased T-helper cell numbers may occur, but this does not have the devastating clinical picture of full-blown AIDS. There may be individuals classified as having early-stage AIDS (pre-AIDS) who show these signs. It is currently not possible to predict which of these will develop the more severe symptoms.

Much of the evidence implicates HTLV-III as the etiological agent of infectious AIDS. First, there is a conclusive epidemiology; more than 95% of patients with AIDS have antibodies specific to HTLV-III. Second, reproducible identification and isolation of virus in this disease has been achieved; more than 100 variants of HTLV-III have been isolated from AIDS patients. Third, transmission of the disease has been observed to normal, healthy individuals who have received blood transfusions containing infected donor blood.

HTLV-III has been shown to share several properties with HTLV-I and HTLV-II, but also to be morphologically, biologically and antigenically distinct. RC Gallo et al., Frequent Detection and Isolation of Cytopathic Retroviruses (HTLV-III) from Patients with AIDS and at Risk for AIDS. Science. 224 : 500-503 (1984). For example, HTLV-III has been shown to be related to HTLV-I and HTLV-II in its antigenic properties. by demonstrating cross-reactivity with antibodies against HTLV-I and HTLV-II core proteins, p24 and p19, and envelope antigens, and by nucleic acid cross-hybridization studies with cloned HTLV-I and HTLV-II DNAs. However, unlike HTLV-I and HTLV-II, it lacks the ability to infect and transform T cells from normal cord blood and bone marrow in vitro, and it exhibits cytopathic effect only on infected cells.

Like the RNA genome of other retroviruses, the RNA genome of HTLV-III contains three genes that encode viral proteins: 1) the gag gene, which encodes the internal structural proteins (nucleocapsid or core); 2) the pol gene, which encodes the RNA-targeting DNA polymerase (reverse transcriptase); and 3) the env gene, which encodes the virion envelope glycoproteins. Furthermore, the HTLV-III genome contains a region called Px, located between the env gene and the 3' LTR, which appears to be involved in the functional killing of the virus.

At present, AIDS is still difficult to diagnose before clinical manifestations appear. At present, no method is available to prevent the disease. Treatment of AIDS patients is generally unsuccessful, and victims suffer from the devastating effects that HTLV-III has on the body.

Summary of the invention

This invention is based on the cloning of HTLV-III DNA in recombinant vector host systems by the applicant that can express immunologically reactive HTLV-III polypeptides. Based on the cloning of HTLV-III DNA in systems expressing immunologically reactive polypeptides, Applicant has developed useful methods for the diagnosis, treatment and prevention of AIDS. Applicant has developed methods for the detection of HTLV-III and antibodies to HTLV-III in body fluids (e.g., blood, saliva, semen) and useful methods for immunotherapy (e.g., vaccination and passive immunization against AIDS). Furthermore, Applicant has developed methods for preparing HTLV-III DNA probes and RNA probes useful for the detection of HTLV-III in body fluids. Using these recombinant DNA methods, polypeptides encoded by a segment of the HTLV-III genome have been produced. The polypeptides encoded by a 1.1 Kb EcoRI restriction fragment from HTLV-III cDNA have been produced. The expressed polypeptides have been isolated. These polypeptides are immunologically reactive with sera from patients with AIDS and with antibodies to HTLV-III, and are therefore useful in testing blood or other body fluids for the presence of antibodies to HTLV-III. Therefore, Applicant's invention provides not only a method for diagnosing AIDS, but also for preventing transmission of the disease to others by blood or blood components harboring HTLV-III. The latter is particularly valuable in testing donor blood before it is transfused or used to produce blood components (e.g., factor VIII for the treatment of hemophilia; factor IX).

The polypeptides produced by the recombinant DNA techniques are used in the production of antibodies, including monoclonal antibodies, against the virus. Such antibodies form the basis of immunoassays and diagnostic techniques for the direct detection of HTLV-III in body fluids, such as blood, saliva, semen, etc. Neutralizing antibodies against the virus can be used to provide passive immunization against the disease.

Applicant's cloning of HTLV-III DNA in such recombinant vector host systems also provides the basis for determining the nucleotide sequence of HTLV-III DNA. The DNA probes are substantially homologous to the 1.1 Kb EcoRI DNA unique to the HTLV-III genome. The DNA probes provide another method for detecting HTLV-III in blood, saliva, or other body fluids. RNA probes containing regions unique to the HTLV-III genome can also be prepared and used to detect HTLV-III in body fluids.

Short description of the characters

Figure 1 shows a representation of the HTLV-III DNA. Figure 1a shows sites where the genome is cleaved by the restriction enzyme SstI, and Figure 1b shows the fragments of the HTLV-III genome generated by the action of the restriction enzymes Kpn, EcoRI and HindIII.

Figure 2 is a representation of HTLV-III DNA. Figure 2a shows the location of restriction enzyme cleavage sites in the genome, and Figure 2b shows the location of DNA inserts in the HTLV-III genome in clones with an open reading frame. (+) and (-) indicate the reactivity and lack of reactivity, respectively, of the fusion proteins expressed by cells transformed with the ORF vectors with sera from AIDS patients.

Figure 3 shows the nucleotide sequence of HTLV-III DNA and the predicted amino acid sequence of the four longest open reading frames. Restriction enzyme cleavage sites are indicated above the nucleotide sequence.

Figure 4 shows an immunoblot showing the position of HTLV-III-env-beta-galactosidase fusion proteins on an SDS-polyacrylamide gel.

Figure 5 shows sites where the genome is cut by the restriction enzyme EcoRI and the construction of the recombinant plasmid containing HTLV-III DNA.

Figure 6 shows an immunoblot showing the positions of peptides on nitrocellulose blots generated from bacterial cells transformed with recombinant constructs ompA1-R-6; ompA2-R-7 and ompA3-R-3 into which a 1.1 Kb EcoRI HTLV-III cDNA restriction fragment had been inserted. Figure 6a shows the nucleotide sequence of the opmA signal peptide and the relevant region of the recombinant plasmids ompA1-R-6; ompA2-R-7 and ompA3-R-3.

Figure 7 shows an immunoblot demonstrating the blocking of the reaction between HTLV-III antigens and an AIDS serum by lysates of E. coli containing the recombinant HTLV-III DNA plasmid ompA1-R-6 (lanes 1 to 5) and showing the lack of blocking of the reaction by lysates of control E. coli cells (lanes 6 to 10).

Figure 8 shows an immunoblot indicating the presence or absence of antibodies in sera from healthy individuals (lanes 1 to 3) and from AIDS patients (lanes 4 to 11) against the peptide encoded by the 1.1 Kb EcoRI HTLV-III restriction fragment of the HTLV-III cDNA. Purified HTLVIII virus (series A) or total cell lysates of the bacterial clone ompA1-R-6 (O1R6) were reacted with the serum samples.

Figure 9 shows the open reading frame expression vector pMR100 containing HTLV-III DNA.

Figure 10 depicts Lambda-CI-HTLV-III-beta-galacosidase fusion proteins. Figure 10a shows an immunoblot showing the position of the Lambda-CI-HTLv-III-beta-galacosidase fusion proteins on an SDS-polyacrylamide gel, and Figure 10b shows the immunological reaction of such proteins with sera from AIDS patients.

Best mode for carrying out the invention

Despite the similarities between HTLV-III and the other virus members of the HTLV bovine leukemia virus (BLV) family, the biology and pathology of HTLV-III differ considerably. For example, only relatively little homology was found in the HTLV-III genome when compared with the HTLV-I or HTLV-II genomes. HTLV-III infection often results in drastic immunosuppression (AIDS) as a consequence of the removal of the OKT4(+) cell population. This effect is a reflection of an enhanced cytopathic rather than a transforming effect of HTLV-III infection on the OKT4(+) cells in in vitro lymphocyte cultures. In contrast, infection with HTLV-I leads to a low incidence of T-cell leukemia lymphomas (an OKT4(+) cell malignancy). There is also evidence of a certain degree of immunodeficiency in HTLV-I patients. Infection of primary lymphocytes in culture by HTLV-I and -II leads to the in vitro transformation of predominantly OKT4(+) cells. A cytopathic effect of HTLV-I infection on lymphocytes is evident, but the effect is not as pronounced as observed for HTLV-III.

HTLV-III also differs from HTLV-I and -II in the extent of infectious virion production in vivo and in vitro. High titres of cell-free, infectious virions can be isolated from the semen and saliva of AIDS patients and the supernatant from cultures infected with HTLV-III. Very few, if any, cell-free infectious virions can be obtained from adult T-cell leukemia lymphoma (ATLL) patients or from cultures infected with HTLV-I or -II.

The envelope glycoprotein is the main antigen recognized by antisera from AIDS patients. In this respect, HTLV resembles other retroviruses, for which the envelope glycoprotein is typically the most antigenic viral polypeptide. Furthermore, neutralizing antibodies are generally directed against the envelope glycoprotein of the retrovirus. 88 to 100% of serum samples from AIDS-infected individuals contain antibodies reactive with antigens of HTLV-III; the main immunological reactivity was directed against p41, the presumed envelope antigen of HTLV-III. Antibodies to core proteins have also been detected in sera from AIDS patients, but these do not appear to be as effective as the presence of antibodies to envelope antigen as an indicator of infection.

Characterization of the p41 antigen of HTLV-III has proven difficult because the viral envelope is partially destroyed during the virus inactivation and purification process. This invention provides an answer to the urgent need to characterize this antigenic component of the HTLV-III virus and to determine the presence and identity of other viral antigenic components by various means. It provides products such as HTLV-III polypeptides, antibodies to the polypeptides, and RNA and DNA probes, as well as methods for their preparation. These serve as the basis for screening, diagnostic and therapeutic products and methods.

This invention relates to HTLV-III polypeptides produced by translating a recombinant DNA sequence encoding HTLV-III proteins. Polypeptides produced in this manner which are immunologically reactive with serum from AIDS patients or antibodies to HTLV-III are referred to as recombinant DNA-produced immunologically reactive HTLV-III polypeptides. They include the polypeptides produced by translating the recombinant DNA sequences located in a 1.1 Kb EcoRI restriction fragment of HTLV-III cDNA.

The polypeptides encoded by this region of HTLV-III can be used in immunochemical tests to detect antibodies to HTLV-III and HTLV-VIII infections. These methods can be useful in the diagnosis of AIDS. They can also be used to test blood before it is used for transfusion or to produce blood components (e.g., factor VIII for the treatment of hemophilia). The availability of testing will reduce the risk of AIDS transmission.

Detection of antibodies reactive with the polypeptides can be performed using a number of established methods. For example, an immunologically reactive HTLV-III polypeptide can be fixed to a solid phase (such as polystyrene beads or other solid support). The solid phase is then incubated with the blood sample to be tested for antibodies to HTLV-III. After an appropriate incubation period, the solid phase and the blood sample are separated. Antibody bound to the solid phase can be detected with labeled polypeptide or with a labeled antibody directed against human immunoglobulin.

The HTLV-III polypeptides can be used in a vaccine to prevent AIDS.

The polypeptides can also be used to produce antibodies, including monoclonal antibodies, against the HTLV-III polypeptides. These antibodies can be used in immunochemical assays for the direct detection of the virus in body fluids (such as blood, saliva, and semen). Tests that use monoclonal antibodies against specific HTLV-III antigenic determinants will reduce false-positive results, thereby improving the accuracy of tests for the virus. Antibodies against the virus may also be useful in immunotherapy. For example, antibodies can be used to passively immunize against the virus.

The methods for producing the polypeptides are also the subject of the present invention, as are diagnostic methods based on these polypeptides.

HTLV-III polypeptides

Genetic engineering techniques are used to isolate a 15 kd peptide encoded by a 1.1 Kb EcoRI-HTLV-III restriction fragment of HTLV-III DNA. These techniques are also used to sequence the fragments encoding the polypeptides. The provirus genes integrated into the host cell DNA are molecularly cloned and the nucleotide sequence of the cloned proviruses is determined.

An E. coli expression library of HTLV-III DNA is prepared. The HTLV-III genome is cloned and then restriction enzymes are used to cut the cloned HTLV-III genome to produce DNA fragments (Fig. 1 and 2).

HTLV-III DNA fragments of approximately 200 to 500 bp are isolated from an agarose gel, end-filled with T4 polymerase and ligated to linker DNA. The linker-ligated DNA is then treated with a restriction enzyme, purified from agarose gel and cloned into an expression vector. Examples of the expression vectors used are: OmpA, pIN (A, B and C), lambda pL, T7, lac, Trp, ORF and lambda gt11. Furthermore, vectors for mammalian cells can be used, such as pSv28pt, pSv2neo, psvdhfr and VPV vectors, and yeast vectors such as GALI and GAL10.

The bacterial vectors contain the Lac coding sequences into which HTLV-III DNA can be inserted to produce β-galactosidase fusion protein. The recombinant vectors are then introduced into bacteria (e.g., E. coli); those cells that take up a vector containing HTLV-III DNA are said to be transformed. The cells are then screened to identify cells that have been transformed and express the fusion protein. For example, the bacteria are plated on NacConkey agar plates to verify the phenotype of the clone. If functional β-galactosidase is produced, the colony appears red.

Bacterial colonies are also screened with HTLV-III DNA probes to identify clones containing the DNA region of interest. Clones that are positive when screened with the DNA probe and positive on the MacConkey agar plates are isolated.

This identification of cells containing the HTLV-III DNA sequences makes it possible to produce HTLV-III polypeptides that react immunologically with an HTLV-III specific antibody. The cells of the selected colonies are maintained in culture under such conditions that that allow the expression of the hybrid protein. The cell protein is then obtained in a conventional manner. For example, the culture can be centrifuged and the resulting cell pellet broken up. Polypeptides secreted by the host cells can be obtained from the cell culture supernatant (without destroying the cells).

Total cellular protein is analyzed using an SDS-polyacrylamide gel electrophoresis run. Fusion proteins are identified at a position on the gel that does not contain any other protein. Western blot analyses are also performed on clones that are positive after screening. Such analyses are performed on serum from AIDS patients with the result that it is possible to identify clones that express HTLV-III-H-galactosidase fusion proteins (antigens) that cross-react with the HTLV-III specific antibody.

Lambda-10 clones carrying the HTLV-III DNA are cloned from the replicating form of the virus. As the retrovirus replicates, double-stranded DNA is produced. The cloned HTLV-III DNA is digested with the restriction enzyme SstI (Fig. 1a). Since there are two SstI recognition sites within the LTR of the HTLV-III DNA, an LTR region is absent from the cloned DNA sequence that was removed from the lambda-10 vector. As a result, a small (approximately 200 bp) fragment of the HTLV-III DNA is missing.

The resulting DNA is linearized and fragments are generated by digesting the linearized genomic DNA comprising the env gene region with the restriction enzyme EcoRI (Fig. 1b). The resulting 1.1 Kb EcoRI-EcoRI fragments are isolated by gel electrophoresis and electroelution. These fragments are randomly sheared to generate smaller fragments. The fragments thus generated are separated from the agarose gel, and DNA fragments between approximately 200 to 500 bp are eluted.

The eluted 200 to 500 bp DNA fragments are filled in at the ends using E. coli T4 polymerase and the blunt ends are ligated into an open reading frame (ORF) of an expression vector such as pMR100. This ligation can be done at the SinaI site of the pMR100 vector, which contains two promoter regions, hybrid coding sequences of the lambdaCI gene and lacI-Lac2 gene fusion sequences. In the vector, these sequences are out of frame; as a result, the vector is not productive. The HTLV-III DNA is inserted into the vector; the correct DNA fragments correct the reading frame, resulting in CI-HTLV-III β-galactosidase fusion proteins being produced. The expression of the hybrid is under the control of the lac promoter. Based on the sequence of pMR100, it appears that if a DNA fragment insert cloned into the SmaI site is to create an appropriate reading frame between the lambdaCI gene fragment and the lac Z fragment, the inserted DNA fragment must not contain any stop codons in the reading frame dictated by the lambdaCI gene frame.

The recombinant pMR100 vectors are then introduced into E. coli. The bacteria are plated on MacConkey agar plates to verify the phenotype of the clones. If functional β-galactosidase is produced, the colony will appear red. The colonies are also screened with HTLV-III DNA probes for the purpose of identifying those clones containing the insert. Clones that are positive after screening with the DNA probe and are positive on the MacConkey agar plates are isolated.

The cells from these selected colonies are cultured. The culture is centrifuged and the cell pellet is broken up. The total cellular protein is analyzed using an SDA polyacrylamide gel run. The fusion proteins are identified at a position on the gel that does not contain any other protein (Fig. 4).

Western blot analyses are also performed on the clones found to be positive. Sera from AIDS patients are used, which thus allow the identification of clones that express HTLV-III-β-galactosidase fusion proteins that cross-react with the HTLV-III specific antibody. A thousand clones were screened using this procedure; 6 were positive.

Because of the nature of the pMR100 cloning vehicle, a productive DNA insert should also be expressed as part of a longer fusion polypeptide. Recombinant clones containing the HTLV-III env gene were identified by colony hybridization. Production of longer fusion polypeptides harboring functional β-galactosidase activity was verified by phenotypic identification on MacConkey agar plates, by β-galactosidase enzyme assays, and by analysis on 75% SDS polyacrylamide gels. Immunological reactivity of the larger proteins with antibodies to HTLV-III was determined by Western blot analyses using serum from AIDS patients. These large fusion proteins also reacted with anti-β-galactosidase and anti-CI antiserum. This finding is consistent with the hypothesis that they represent proteins of CI-HTLV-III-LacIZ.

The insert fragment of HTLV-III with the open reading frame is further analyzed by DNA sequence analysis. Since one of the two BamHI sites that encode the SmaI cloning site in pMR100 flank is destroyed during the cloning step, positive clones are digested with the restriction enzymes HindIII and ClaI to release the introduced HTLV-III DNA fragment. The HTLV-III ORF inserts are isolated from the fusion recombinant and cloned into N13 cloning vectors mp18 and mp19 digested with HindIII and AccI for sequencing. DNA sequences of the positive ORF clones are then determined.

Fragments of HTLV-III DNA of approximately 200 to 500 bp are then isolated from the agarose gel, end-filled with T4 polymerase, and ligated to EcoRI linkers. The DNA ligated to the EcoRI linkers is then treated with EcoRI, purified from the 1% agarose gel, and cloned into an expression vector, lambda gt11. This vector contains lacZ gene encoding sequences into which the foreign DNA can be inserted to generate β-galactosidase fusion protein. Expression of the hybrid gene is under the control of the Lac repressor. The Lac repressor gene, lacI, is contained on a separate plasmid pMC9 in the host cell, E. coli Y1090. Serum from AIDS patients was used to screen the lambdagt11 library of HTLV-III genomic DNA containing 1.5 x 104 recombinant phages. By screening 5000 recombinants, 100 independent clones were isolated that produced strong signals. The positive recombinant DNA clones were further characterized for their specific gene expression. Rabbit hyperimmune serum against P24 was also used to identify the gag gene-specific clones. Nick-translated DNA samples of specific HTLV-III genes, particularly the gag gene, env gene and Px gene, were used to group the positive immunoreactive clones into specific gene regions.

Recombinant clones that gave strong signals with AIDS serum and contain insert DNA encoding the HTLV-III gag, pol, -sor and -env-lor gene regions were extensively investigated by mapping the inserts with restriction enzymes and by DNA sequence analysis.

Determining the nucleotide sequence of HTLV-III DNA

Genetic engineering techniques are used to determine the nucleotide sequence of HTLV-III DNA. One technique that can be used to determine the sequence is a shotgun/random sequencing method. HTLV-III is randomly sheared into fragments approximately 300 to 500 bp in size. The fragments are cloned, e.g., using m13, and the colonies are screened to identify those with an HTLV-III DNA fragment insert. The nucleotide sequence is then generated, with multiple analyses generating overlaps in the sequence. Both strands of HTLV-III DNA are sequenced to determine the orientation. Restriction mapping is used to verify the sequence data generated.

The nucleotide sequence of a cloned HTLV-III genome (BH10) is shown in Fig. 3, in which the position of u-protein p17 coding sequences and the N-terminus of gag p24 and the C-terminus of gag p15 (which overlaps with the N-terminus of the Mol protein) are indicated. The open reading frames (ORFs) for pol, sor, and env-lor are also indicated. The sequence of the remaining 182 bp of HTLV-III DNA not present in clone BH10 (including part of R, U5, the tRNA primer binding site, and part of the leader sequence) was obtained from clone HXB2. The sequences of the additional clones (BH8 and BH5) are also shown. Restriction enzyme cleavage sites are listed above the nucleotide sequence; in clone BH8, but not in clone BH10, present sites are in parentheses. Deletions ([]) at nucleotides 251, 254, 5671 and 6987 to 7001 are indicated. Nucleotide positions (to the right of each line) begin with the transcription initiation site. Amino acid residues are numbered for the four longest open reading frames, in each case beginning after the preceding termination codon, except for gag, which is numbered from the first methionine codon (to the right of each line). A proposed peptide cleavage site (V) and possible asparagine-associated glycosylation sites (*) are shown for the env-lor open reading frame. The sequences in the LTR derived from clones BH8 and BH10, listed at the beginning of the figure, are from the 3' portion of each clone and are presumably identical to those in the 5' LTR of the integrated copies of these viral genomes.

Clone HXB2 was derived from a recombinant phage library of XbaI-digested DNA from HTLV-III-infected H9 cells, cloned into lambdaJ1. H9 cells are human leukemia cells infected with a pool of HTLV-III from blood of AIDS patients, F. Wong-Staal, Nature, 321 November 1984. Clones of the cloning vector BH10, BH8 and BH5 were derived from a library of SstI-digested DNA from the Hirt supernatant of HTLV-III-infected H9 cells, cloned into lambdagtWes. lambdaB. Both libraries were screened with cDNA probes synthesized from virion RNA using oligo.dT as a primer. Clones BH8, BH5 and parts of HXB1 were sequenced as described by Maxam and Gilbert (1980) Maxam, A.M. and Gilbert, Co. Methods in Enzymology. 65 : 499 to 560. Clone BH10 was sequenced by the method of Sanger modified by the use of oligonucleotides complementary to the M13 insert sequence as primers and using the Klenow fragment of DNA polymerase I or reverse transcriptase as polymerase.

Preparation of RNA, RNA probes and DNA probes specific for HTLV-III

DNA sequences representing an entire gene or a segment of a gene of HTLV-III are inserted into a vector, such as a T7 vector. In this embodiment, the vector has the Tceu promoter from the T cell gene 10 promoter and DNA sequences encoding eleven amino acids of the T cell gene 10 protein.

The vectors are then used to transform cells such as E. coli. The T7 vector uses the T7 polymerase, which catalyzes RNA formation and recognizes only the T7 promoter, which is the site where RNA polymerases bind to initiate transcription. The T7 polymerase does not recognize the E. coli promoter. As a result, the T7 vector directs the production of RNA that is complementary to the HTLV-III DNA insert when HTLV-III DNA sequences are inserted behind the promoter and polymerase genes of the T7 vector that recognize them to the exclusion of other signals and a terminator are placed immediately behind the HTLV-III DNA sequences.

Determination of the nucleotide sequence of HTLV-III DNA also provides the basis for the preparation of DNA probes. Both RNA probes and DNA HTLV-III probes must contain characteristic regions of the HTLV-III genome to be useful for detecting HTLV-III in body fluids. There is relatively little homology between the HTLV-III genome and the HTLV-I and II genomes, and the probes contain regions that are characteristic of HTLV-III (i.e., not shared with HTLV-I or II).

Viral RNA or DNA can be used to detect HTLV-III in, for example, saliva, which is known to contains very high concentrations of the virus. This can be done, for example, using a dot blot, where the saliva sample is denatured, applied to paper and then examined with one of the probes. If saliva is used as the test fluid, the detection of HTLV-III is much faster and easier than in the case of blood testing.

Production of monoclonal antibodies reactive with the HTLV-III polypeptides

Monoclonal antibodies reactive with HTLV-III polypeptideb are produced by antibody-producing cell lines. The antibody-producing cell lines may be hybridoma cell lines, commonly known as hybridomas. The hybrid cells are formed by fusion of cells that produce an antibody against the HTLV-III polypeptide and an immortalizing cell, i.e., a cell that provides the hybrid cell with long-term stability as a tissue culture. In producing the hybrid cell lines, the first fusion partner - the antibody-producing cell - may be a spleen cell from an animal that has been immunized against the HTLV-III polypeptide. Alternatively, the antibody-producing cell may be an isolated B cell that produces antibodies against an HTLV-III antigen. The lymphocytes may be obtained from the spleen, peripheral blood, lymph nodes, or other tissue. The second fusion partner - the immortalized cell - can be a lymphoplastoid cell or a plasmacytoma cell, such as a myeloma cell, which itself is an antibody-producing cell but also malignant.

Mouse hybridomas that produce monoclonal antibodies against HTLV-III polypeptides are produced by the fusion of mouse myeloma cells and spleen cells from mice immune to the polypeptide. A variety of different immunization protocols can be followed to immunize the mice. For example, the mice can receive primary and booster immunizations with the purified polypeptide. Fusions are performed using standard procedures (Köhler and Milstein, (1975) Nature (London) 256. 495 to 497; Kennet, R., (1980) in Monoclonal Antibodies (Kennet et al., editors, pages 365 to 367, Plenum Press, New York)

The hybridomas are then tested for the production of antibodies that are reactive with the polypeptide. This can be done using test methods known in the art.

Another way to produce antibody-producing cell lines is to transform antibody-producing cells. For example, a B lymphocyte obtained from an animal immunized against the HTLV-III polypeptide can be infected with a virus, such as the Epstein-Barr virus in the case of human B lymphocytes, and transformed to yield an immortalized antibody-producing cell. See, e.g., Kozbor and Rodor (1983) Immunology Today 4 (3), pages 72 to 79. Alternatively, the B lymphocyte can be transformed by a transforming gene or a transforming gene product.

The monoclonal antibodies against the HTLV-III polypeptide can be produced in large quantities by injecting the antibody-producing hybridomas into the peritoneal cavity of mice and, after an appropriate period of time, isolating the ascites fluid containing very high titres of homogeneous antibody and isolating the monoclonal antibody therefrom. Xenogeneic hybridomas should be irradiated or athymic nude mice. Alternatively, the antibodies can be produced by culturing cells that produce the HTLV-III polypeptide in vitro and isolating the secreted monoclonal antibodies from the cell culture medium. The antibodies produced by these methods can be used in diagnostic assays (e.g., detection of HTLV-III in body fluids) and in passive immunotherapy. The antibodies reactive with the HTLV-III polypeptides form the basis for diagnostic tests to detect AIDS or the presence of HTLV-III in biological fluids (e.g., blood, semen, saliva) and for passive immunotherapy. For example, it is possible to produce anti-p41, attach it to a solid phase using conventional techniques, and contact the body fluid to be tested with the immobilized antibody. In this way, HTLV-III (antigen) can be detected in the body fluid; This procedure results in far fewer false-positive test results than tests that detect antibodies against HTLV-VIII.

The invention is further explained below using examples.

EXAMPLE 1 Production of sonicated DNA fragments

10 µg of gel-purified HTLV-III restriction fragments were sonicated to an average fragment size of 500 bp. After sonication, the DNA was passed through a DEAE-cellulose column in 0.1 TBE to reduce the volume. The DEAE-bound DNA was washed with 5 ml of 0.2 M NaCl-TE (2 M NaCl, 10 mM Tris HCl pH 7.5, 1 mM EDTA) and then eluted with 1 M NaCl-TE and precipitated with ethanol. The size distribution of the sonicated DNA was then on a 1.2% agarose gel. DNA fragments of the desired length (200 to 500 bp) were then eluted from the gel. T4 DNA polymerase was then used to fill in and/or truncate the single-stranded DNA ends generated in the sonication procedure. The DNA fragments were incubated with T4 polymerase in the absence of added nucleotides for 5 minutes at 37°C to remove nucleotides from the 3' end, then all 4 nucleotide precursors were added to a final concentration of 100 µM and the reaction mixture was incubated for an additional 30 minutes to repair the single strands hanging over at the 5' end. The reaction was stopped by heat inactivation of the enzyme at 68°C for 10 minutes. The DNA was extracted once with phenol, precipitated with ethanol and resuspended in TE.

EXAMPLE 2 Cloning randomly sheared DNA fragments

The sonicated blunt-ended HTLV-III DNA fragments were ligated into the SmaI site of the ORF expression vector pMR100 and transformed into host LG90 cells using conventional transformation procedures. The β-galactosidase positive phenotype of the transformants was identified by plating the transformed cells on McConkey agar plates containing ampicillin (25 µg/ml) and examining the phenotype after 20 hours at 37°C.

EXAMPLE 3 Analysis of hybrid proteins

Ten milliliter samples of cells from a saturated overnight culture grown in L-medium containing ampicillin (25 ug/ml) were centrifuged, the Cell pellet was resuspended in 500 µl of 1.2-fold concentrated Laemmli sample buffer. Cells were resuspended by vortexing and boiled for 3 minutes at 100ºC. The lysate was then repeatedly passed through a 22-gauge needle to reduce the viscosity of the lysate. Approximately 10 µl of protein samples were electrophoresed on 7.5% SDS-PAGE (SDS-polyacrylamide) gels.

Electrophoretic transfer of proteins from the SDS-PAGE gels to nitrocellulose paper was carried out according to Towbin et al. (Proc. Natl. Acad. Sci., USA, in, 1979, 4350 ff). After transfer, the filter was incubated for 2 hours at 37°C in a solution of 5% (w/v) nonfat milk in PBS containing 0.1% antifoam A and 0.0001% merthiolate to saturate all available protein binding sites. Reactions with AIDS antisera were carried out in the same milk buffer containing 1% AIDS patient antisera preabsorbed with E. coli lysate. Reactions were carried out in a sealed plastic bag at 4°C for 18 to 24 hours on a rotary shaker. Following this incubation, the filter was washed three times for 20 minutes each at room temperature in a solution containing 0.5% deoxycholic acid, 0.1% M NaCl, 0.5% Triton X-100, 10 mM phosphate buffer pH 7.5 and 0.1 mM PMSF.

To visualize antigen-antibody interactions, the nitrocellulose was then incubated with the second goat anti-huinan antibody that had been iodinated with 125I. The reaction with the iodinated antibody was carried out at room temperature for 30 minutes in the same milk buffer used for the first antibody. The nitrocellulose was then washed as previously described and exposed at -70°C using Kodak XARS film with an intensifying screen.

EXAMPLE 4 Screening the HTLV-III ORF library using colony hybridization

E. coli LG90 transformants were screened with HTLV-III DNA probes containing the DNA regions of interest (e.g., HTLV-III gag, env, or PX gene-specific sequences). Colonies were grown on nitrocellulose filters and screened according to the method of Grunstein and Hogness using nick-translated HTLV-III DNA as a hybridization probe.

The DNA fragment was generally excised by restriction endonuclease digestion, gel purified and 32P-labeled to a specific activity of 0.5·10⁸ cpm/ug by nick translation (Rigby, P.W.J. et. al., Journal Molecular Biology 113, 237 (1977)). Duplicate nitrocellulose filters with DNA attached were prehybridized with 6 SSC (0.9 M NaCl/0.09 M sodium citrate, pH 7.0), 5 Denhardt's solution (Denhardt's solution: 0.02% each of polyvinylpyrrolidone, Ficoll and bovine serum albumin), 10 µg of denatured sonicated E. coli DNA per ml at 55°C for 3 to 5 hours. The filters were then placed in a fresh sample of the same solution to which the denatured hybridization probe had been added. Hybridization was allowed at 68°C for 16 hours. The filters were repeatedly washed in 0.3 SSC at 55°C and then exposed to X-ray film.

EXAMPLE 5 Peptide of HTLV-III produced using recombinant DNA that is immunologically reactive with sera from patients with AIDS

An expression vector, pIN-III-ompA (ompA), was used.

ompA possesses the lipoprotein (the most abundant protein in E. coli) gene promoter (lpp) and the lacUV5 promoter operator (Fig. 5). ompA vectors also contain the DNA segment encoding the lac repressor, which allows the expression of the inserted DNA to be regulated by inducers of the lac operon, such as IPTG. The ompA cloning vehicles contain three unique restriction enzyme sites EcoRI, HindIII, BamHI in all three reading frames and allow the insertion of the DNA into any of these restriction sites.

Various restriction fragments were excised from the recombinant clone lambdaBH10, which contains a 9 Kb HTLV-III DNA insert in the SstI site of the vector lambdagtWES-lambdaB. These restriction fragments were then inserted into the opmA vectors in all three reading frames and used to transform E. coli JA221 cells. The transformants were first screened for HTLV-III DNA using in situ colony hybridization and the use of nick-translated HTLV-III DNA probes. The positive clones were then screened for expression of HTLV-III antigenic peptides using HTLV-III-specific antibodies. For this purpose, lysates of E. coli cells containing HTLV-III DNA recombinant plasmids were electrophoresed on 12.5% SDS-polyacrylamide gel and electroblotted onto nitrocellulose filters. The filters were then incubated first with well-characterized sera from AIDS patients and then with 125I-labeled goat anti-human IgG antibodies. The washed filters were autoradiographed to identify peptides reactive with anti-HTLV-III antibodies.

Several gene segments were identified that encode peptides that induce an immune response with anti-HTLV-III antibodies Among these is a 1.1 Kb EcoRI restriction fragment. This fragment was inserted into opmA vectors in all three reading frames (Fig. 5). Cells were grown at 37°C in L medium containing 100 mg/ml ampicillin to an OD₆₀₀ of 0.2. At this time, cell cultures were divided into two aliquots. IPTG was added to one aliquot at a final concentration of 2 mM (induced). IPTG was not added to the other aliquot (uninduced). Upon IPTG induction, all three plasmid constructs (designated ompA₁-R-6 (O1R6), ompA₂-R-7 (O2R7), and ompA₃-R-3 (O3P3)) produced a 15 Kd peptide that reacts strongly with anti-HTLV-III antibodies in sera from AIDS patients (Fig. 6, lane 1, purified HTLV-III virions; lanes 2 and 3, O1R6 uninduced and induced; lanes 4 and 5, O2R7 uninduced and induced; lanes 6 and 7, O3R3 uninduced and induced). This reactivity is not detected when sera from normal individuals are used.

DNA sequence data from the HTLV-III genome indicate that there is an open reading frame within the pol gene located at the 5' end of the EcoRI fragment. DNA sequence analyses of the three recombinant constructs O1R6, O2R7 and P3R3 confirmed that each of these recombinants has a different reading frame of the HTLV-III plus strand linked to the coding sequence of each vector. Only in O3R3 is the reading frame of the inserted DNA in phase with that dictated by the signal peptide in the ompA vector; in O1R6 and O2R7 the DNA of the pol gene segment is out of phase (Fig. 6a).

A 6 bp ribosome binding site, AAGGAG (Shine-Dalgarno sequence), is located at nucleotide positions 24 to 29, and an initiation codon, ATG, is located 11 bp downstream (positions 41 to 43). The The 15 Kd peptide synthesized appears to be translated from the transcripts using this internal start codon. If this is true, the peptide begins at the ATG located at positions 41 to 43 and ends at the stop codon at positions 446 to 448, producing a peptide of 135 amino acid residues encoded by the 3'-end segment of the pol gene of HTLV-III.

In addition to the 15 Kd peptide, the O3R3 construct, in which the reading frame of the HTLV-III DNA pol gene is in phase with that provided by the vector, produces two additional peptides of approximately 19 Kd and 16.5 Kd in size (Fig. 6). It is possible that the 19 Kd peptide contains an additional 35 amino acid residues, 21 of which are from the signal peptide encoded by the ompA3 vector and 14 are encoded by the inserted HTLV-III DNA itself. The 16.5 Kd peptide may represent the processed 19 Kd peptide with the signal peptide cleaved.

The O1R6 and O2R7 constructs also produce another peptide of approximately 17.5 Kd (Fig. 6) that reacts weakly with sera from AIDS patients. The origin of this peptide is unclear. The 1.1 Kb EcoRI fragment contains a second potential coding region, termed the short open reading frame (SOR), which extends from nucleotide position 360 to 965 (Fig. 5). Four of the five AUG methionine codons in this region are located close to the 5' end of this open reading frame. This DNA segment could encode peptides of 192, 185, 177, or 164 amino acid residues. However, there is no clearly recognizable ribosome binding site at the 5' end of this open reading frame.

There is further evidence to support the conclusion that the 15 Kd peptide is indeed derived from the pol gene. First, the deletion of the 3' end of the StuI-EcoRI fragment from the 1.1 Kb EcoRI insert of O1R6, O2R7 and O3R8 (Fig. 5) does not affect the synthesis of the 15 Kd peptide. Second, clones containing only the 5' end, the EcoRI-NdeI fragment, nevertheless produce the same 15 Kd peptide. Finally, several recombinant clones containing various DNA fragments with the SOR coding sequence correctly inserted into the open reading frame of the cloning vector pMR100 produced LambdaCI-HTLV-III-β-galactosidase triple fusion proteins that have very low immunological reactivity with anti-HTLV-III antibodies in sera from AIDS patients.

Significant immunological reactivity against the 15 Kd peptide derived from the viral pol gene was demonstrated with sera from AIDS patients. The identity of this immunologically reactive peptide with respect to the banding pattern of HTLV-III virion antigen in SDS-polyacrylamide gel electrophoresis was determined by a competition-inhibition immunoassay. Purified HTLV-III virions were treated with SDS, electrophoresed and electroblotted onto nitrocellulose. Identical filter strips containing disrupted HTLV-III virions were incubated with well-characterized serum from an AIDS patient in the presence or absence of lysates of O1R6, O2R7 or clones of control bacteria. The specific immune reaction between the anti-HTLV-III antibodies present in the AIDS patient's sera and the blotted virion proteins was visualized using 125I-labeled goat antihuman antibody. As shown in Fig. 7, lysates of O1R6 block the immunological reactivity of the viral p31 protein with the AIDS serum, whereas lysates of control cells do not. This result suggests that the recombinant 15 Kd peptide encoded by the 3' end of the viral pA gene also represents part of another virion protein, p31, in the Contrary to the view shared by some that p31 is a cellular protein that is co-purified with the HTLV-III virions.

The widespread presence of antibodies to the 15 Kd peptide in the sera of AIDS patients was also determined. A panel of sera from AIDS patients and normal healthy individuals were tested in Western blot analyses using the lysate of O1R6 as the antigen source. All 20 AIDS sera and none of the 8 normal controls reacted with the 15 Kd peptide. Representative results are shown in Fig. 8. These data indicate that most, if not all, AIDS patients produce antibodies to the viral p31 protein.

Claims (20)

1. An HTLV-III polypeptide expressed by cells transformed with a recombinant vector containing HTLV-III DNA, said polypeptide being immunologically reactive with sera from individuals with acquired immunodeficiency syndrome or sera with antibodies to HTLV-III; wherein said HTLV-III DNA (a) is an EcoRI restriction fragment of about 1.1 Kb and has a nucleotide sequence extending from about nucleotide 4228 to about nucleotide 5327 of said HTLV-III DNA as shown in Figure 3; or (b) is an equivalent of said DNA which encodes an immunologically functional equivalent of said polypeptide. 2. Isolated DNA according to claim 1 (a) or (b) which encodes a polypeptide according to claim 1. 3. A recombination vector containing HTLV-III DNA according to claim 1 (a) or (b) and capable of expressing a polypeptide according to claim 1 after insertion into a host cell. 4. pMR 100 vector containing HTLV-III DNA according to claim 1 (a) or (b) and, after insertion into a host cell, is capable of expressing a polypeptide according to claim 1. 5. A hybrid protein containing a polypeptide according to claim 1 linked to an indicator polypeptide such as beta-galactosidase. 6. A DNA probe containing a DNA sequence which is substantially homologous to the DNA encoding a polypeptide according to claim 1 (a) or (b). 7. A process for producing a polypeptide according to claim 1, comprising the following steps: (a) cutting the HTLV-III DNA to produce DNA fragments; (b) inserting the DNA fragments into an expression vector to produce a recombination vector; (c) transforming a suitable host cell with the recombination vector; and (d) culturing the transformed host cell under conditions suitable for expression of the polypeptide encoded by the inserted HTLV-III DNA. 8. The method of claim 7, wherein the cutting step includes: (a) cutting the HTLV-III DNA using restriction endonucleases to produce restriction fragments of the DNA or (b) Shearing of HTLV-III DNA to produce DNA fragments. 9. The method of claim 7 or claim 8, wherein the expression vector is pMR 100. 10. A monoclonal antibody specific to a polypeptide according to claim 1. 11. Immunoassay for the detection of HTLV-III, in which antibodies according to claim 10 are used. 12. A sandwich immunoradiometric assay for the detection of HTLV-III, which comprises an immobilized antibody according to claim 10 which reacts with HTLV-III polypeptide, and a soluble antibody according to claim 10 which reacts with HTLV-III polypeptide. 13. Test system containing an antibody according to claim 10 which specifically reacts with HTLV-III polypeptide bound to a solid phase and a labeled soluble antibody according to claim 10 which specifically reacts with HTLV-III polypeptide. 14. In vitro method for the detection of antibodies against HTLV-III in body fluid, comprising the following steps: (a) contacting an immunoadsorbent comprising the polypeptide of claim 1 and bound to a solid phase with a body fluid until antibodies to HTLV-III polypeptide in the body fluid bind to the polypeptide of the solid phase; (b) separating the immunoadsorbent from the body fluid; (c) contacting the immunoadsorbent with labeled polypeptide according to claim 1 or labeled antibody against human immunoglobulin; and (d) determining the amount of labelled polypeptide bound to the immunoadsorbent as a detection of antibodies against HTLV-III. 15. System for detecting the presence of antibodies against HTLV-III in body fluids, comprising: (a) an immunoadsorbent containing HTLV-III polypeptide according to claim 1 bound to a solid phase; and (b) labeled HTLV-III polypeptide according to claim 1 or a labeled antibody against human immunoglobulin. 16. A method for detecting HTLV-III nucleic acid in body fluid (e.g. a cell lysate), comprising the following steps: (a) adsorbing the nucleic acid contained in body fluid on an adsorbent; (b) denaturing the adsorbed nucleic acid; (c) contacting the adsorbed nucleic acid with an HTLV-III DNA probe according to claim 6; and (d) Determine whether the probe hybridizes with the adsorbed nucleic acid. 17. A hybridoma cell line producing antibodies specific to an HTLV-III polypeptide according to claim 1. 18. Polypeptide according to claim 1 for therapeutic use, e.g. for vaccination. 19. Use of monoclonal antibodies according to claim 10 for the manufacture of a medicament for use in immunotherapy of acquired immunodeficiency syndrome. 20. Use of a polypeptide according to claim 1 for the manufacture of a medicament for use in vaccination against acquired immunodeficiency syndrome.